Synthesis and in vitro antitumor evaluation of some new thiophenes and thieno[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.09.022

New thiophene (2–13) and thienopyrimidine (15–27) derivatives have been synthesized. Twenty three compounds were screened against five cell lines namely; hepatocellular carcinoma (liver) HepG-2, epidermoid carcinoma (larynx) Hep-2, mammary gland (breast)

Full text of DOI:10.1016/j.bioorg.2018.09.022

Accepted Manuscript
Synthesis and in vitro antitumor evaluation of some new thiophenes and thie-
no[2,3-d]pyrimidine derivatives
Mahasen M.Fouad, Eman R. El-Bendary, Ghada M. suddek, Ihsan A. Shehata,
Mohamed M.El-Kerdawy
PII:
S0045-2068(18)30733-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.022
YBIOO 2519
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
24 July 2018
10 September 2018
11 September 2018
Please cite this article as: M. M.Fouad, E.R. El-Bendary, G.M. suddek, I.A. Shehata, M. M.El-Kerdawy, Synthesis
and in vitro antitumor evaluation of some new thiophenes and thieno[2,3-d]pyrimidine derivatives, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.022
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Synthesis and in vitro antitumor evaluation of some new
thiophenes and thieno[2,3-d]pyrimidine derivatives
Mahasen M.Fouada*, Eman R. El-Bendarya,b, Ghada M. suddekc, Ihsan
A. Shehataa, Mohamed M.El-Kerdawya
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Horus University, Egypt.
cDepartment of Pharmacology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.
*Corresponding author ; E-mail address: sonas_sonas_91@yahoo.com (Mahasen M.
Fouad) Tel: +20502367574
Keywords: Thiophenes, Thienopyrimidines, Synthesis, Antitumor evaluation.
Abstract
New thiophene(2-13) and thienopyrimidine (15-27) derivatives have been synthesized.
Twenty three compounds were screened against five cell lines namely; hepatocellular
carcinoma (liver) HepG-2, epidermoid carcinoma (larynx) Hep-2, mammary gland
(breast) MCF-7, human prostate cancer PC-3 and epitheloid cervix carcinoma HeLa. The
results revealed that compounds 15,16,17,24 and 25 showed the highest antitumor
activity against all tested cell lines compared to Doxorubicin. In order to explain the
expected mode of action of the observed anticancer activity, compounds 15,16,17,24 and
25 were selected to screen their DNA binding affinity and enzyme inhibitory activity
against DNA polymerase, thymidylate synthase and tyrosine kinase. The results revealed
that the tested compounds showed good DNA binding affinity as well as good inhibitory
activity against the three enzymes which might explain the observed anticancer activity
of the target compounds.
1. Introduction
Cancer is defined as large groups of diseases in which cells abnormally divide invading
the body tissues and organs and eventually spreading throughout the body[1].Cancer is a
fatal disease caused over 8.7million deaths globally in 2015 taking the second place after
cardiovascular diseases as leading causes of death[2].
1

Till now there is no practical and completely effective drugs or ways to control the
unstopping progress of this disease. Therefore, there is a continuous global efforts to
identify novel effective, selective and less toxic anticancer agents[3, 4]
.
Thiophenes have been reported to have many types of biological activities, where many
of them used as theraputic agents as antimicrobial[5-7], anticancer[8-10],anti-
inflammatory[11] and antiviral agents[12].Also, it was reported that thienopyrimidines have
promising anticancer activity[13-16] with different mechanisms as it may act as inhibitors
for thymidylate synthase [17] and protien kinase [18-22].Moreover, compounds containing
morpholine and piperazine moieties have been reported to posses good anticancer
activity[23]
.
In the light of the above considerations, we synthesized new thiophene derivatives
bearing morpholine and piperazine moieties in addition to new thieno[2,3-d]pyrimidin-
4(3H)-one derivatives. The new compounds were evaluated for anticancer activity.
DNA-binding affinity and enzyme inhibitory action were also evaluated on the active
derivatives in order to inspect their preliminary mode of action.
2.Results and discussion
2.1. Chemistry
Synthesis of ethyl 5-acetyl-2-amino-4-methylthiophene-3-carboxylate 1 was prepared via
multicomponent condensation between acetyl acetone, ethyl cyanoacetate, sulfur and
diethyl amine in absolute ethanol according to reported procedure[24].The N-acylation of
amino group of compound 1 by using chloroacetyl chloride and chloropropionyl chloride
in DMF in the presence of potassium carbonate yielded compounds 2 and 3 respectively.
In the same manner using 4-florobenzoyl chloride, 4-methoxy benzoyl chloride, p-
toluoyl chloride and 2-thiophenecarbonyl chloride in chloroform in the presence of
triethyl amine yielded compounds 4-7 respectively. The structures of compounds 2-7
1
were established on the basis of the spectral data. For example, the H NMR spectra of
compound 3 showed characteristic triplet-quartet pattern for ethyl ester; the triplet
appeared at 1.4 ppm while the quartet appeared at 4.4 ppm. In addition to two new
triplets of (-CH2CH2Cl) at 3.0 and 3.9 ppm. The neuclophilic substitution of alkyl halides
2

with amines was reported in several publications[25-28], in the present study compounds 8-
13 were obtained through refluxing mixture of chloroacetamido derivative 2 or chloro
propanamido derivative 3 with phenyl piperazine, benzyl piperazine and morpholine in
DMF in the presence of triethyl amine. The structures of these compounds were based on
the basis of their spectral data. For example, in the 1H NMR spectra of compounds 8, 9,
11and 12 the piperazine protons appeared at the expected regions (Experimental part ). In
addition to a singlet signal for( benzylic- CH2) at 3.4-3.6 ppm in case of compounds 9
and 12. Regarding compounds 10 and 13, the 1H NMR spectra showed characteristic two
triplets signals of morpholine ring at 2.6 and 3.7-3.8 ppm.
scheme 1: the synthesis of compounds 2-14
3

In this study, it was unexpected surprise to obtain compound 14 instead of 14a or 14b
when compound 2 or compound 3 was treated with hydrazine hydrate. The reaction were
thought to proceed on either pathway (a) where the neuclophilic attack occurs on the
carbonyl carbon of ester group and results in the formation of the corresponding acid
hydrazide 14a or pathway (b) where the attack takes place on chloromethyl group
associated with elimination of HCl to produce 14b. Although, the neuclophilic
substitution reaction on C-2 of amino thiophene is considered a difficult reaction due to
the electron donation effect of the amino group, once the ring activated by electron
withdrawing substitution at C-3 such as carboxamido[29] or ester groups it becomes
activated towards neuclophilic attack on C-2 .In a similar way, it was reported that the
aminoester thiophene underwent substitution reaction with aniline[30] and this could be
the reasonable explanation in our case. The structure of compound 14 was assigned
1
according to the spectral analysis, as the H NMR showed the triplet-quartet pattern of
the ethyl group in addition to two D2O exchangeable singlet signals at 5.2 and 6.2 ppm
for the NH and NH2 protons, on the other hand the singlet peak of -CH2Cl and the two
triplets of -CH2CH2Cl of compounds 2 and 3 respectively were disappeared from the
spectra . Also, the two nitrogen atoms of hydrazino group appeared as two signals at 200
and 406 ppm in the 15N NMR spectra.
On the other hand, treatment of compound 1 with phenylisothiocyanate, 4-chloro phenyl
isothiocyanate and benzyl isothiocyanate in DMF in the presence of sodium hydroxide
followed by acidification with diluted acetic acid afforded thienopyrimidines derivatives
15-17. Compounds 15 and 17 reacted with hydrazine hydrate in pyridine to give the
corresponding hydrazino derivatives 18 and 19. The structure of the compounds was
established on the basis of the spectral data. For example, the 1H NMR of compounds 15-
17 revealed the disappearance of the two signals of ester group, in addition to the
4

appearance of the aromatic protons of phenyl and benzyl moieties at their expected
region (Experimental part ).
scheme 2: synthesis of compounds 15-17
The N,N-dimethyl formamide dimethyl acetal known as DMFDMA is a very useful
reagent for the synthesis of various heterocyclic compounds. Several procedures have
been reported for the condensation of DMFDMA with carbonyl compounds to yield the
[31,
corresponding enaminone
32].. It was also reported that DMFDMA can form
formamidine derivatives by reacting with amines[33]. In this study, several conditions
were tried including the reported procedure by Prasad et al.[34] to synthesize compound
20a but only compound 20 was obtained when compound 1 was treated with DMFDMA.
The formamidine derivative 20 underwent acidic hydrolysis by using glacial acetic acid
to afford formmamide derivative 21 which was also obtained through treatment of
compound 1 with formic acid in the presence of ammonium acetate. The product
obtained from the two approaches was found to be identical with respect to melting point,
thin layer chromatography (TLC) and spectral data. Another function of DMFDMA is
methylation of sulfur and nitrogen [35], so when compound 15 refluxed with DMFDMA
gave rise to S,N-dialkylated derivative 22. The structure of these compounds was
1
established on the basis of their spectral data. For example, the H NMR spectra of
compound 20 showed singlet peak at 3.3 ppm of ((CH3)2 N-) and another singlet peak at
1
7.7 ppm of (-N=CH-) which both disappeared in the H NMR spectra of compound 21,
instead it showed two new singlet peaks at 8.6 and 11.6 ppm for (-NHCHO) and (-
NHCHO).
5

Several synthetic procedures have been reported for preparation of chalcones[36-38].In this
study chalcones 23-27 were synthesized by the reaction between compound 15 and
appropriate aldehyde in two reaction conditions ethanol/10% NaOH solution[39] and in
sodium ethoxide solution. The structure of the compounds was established on the basis of
1
their spectral data. The HNMR showed two new doublet peaks at 7.1-7.4 and 7.5-7.9
ppm characteristic for the protons of alkene as well as the disappearance of the singlet
peak of protons of acetyl group at 2.5ppm.
scheme 3: Synthesis of compounds 20-22
6

scheme 4: Synthesis of compounds 23-27
7

2.2.Target prediction
Target identification plays a central role in the drug design process. There are many
online open access resources for target hunting in drug discovery such as BindingDB,
DrugCentral and Pharmmapper. So, we tried to investigate the potential targets of our
newly synthesized compounds using Pharmmapper, which is a freely accessed web-
server designed to identify potential target candidates for the given probe small molecules
(drugs, natural products, or other newly discovered compounds with binding targets
unidentified) using pharmacophore mapping approach[40].The web server ranked the top
300 potential targets for each compound based on the binding affinity to the target
compounds. Six targets, involved in cancer therapy are common between the tested
compounds. The fit scores with the top six targets are listed in Table 1
Table 1:The fit scores of the tested compounds with top six targets demonstrated by pharmmapper
Compound
NO.
DNA
polymerase
(1T8E)
Insulin
like
growth
factor 1
receptor
(1IGR)
Tyrosine
Protein
Kinase
HCK
Alk
tyrosine
kinase
receptor
(2YS5)
Thymidylate
synthase 1
(1BSP)
Thymidylate
synthase
(1TVV)
(2C0O)
6.0
5.9
5.9
5.9
5.9
5.8
5.7
5.8
5.8
6.1
5.9
5.9
5.2
5.2
5.7
5.3
5.1
5.9
5.9
5.9
6.0
5.9
5.9
5.9
5.8
6.4
5.9
6.5
5.1
5.2
5.8
5.9
5.8
5.8
5.8
5.6
5.6
5.6
5.9
5.9
5.7
5.7
5.6
6.0
-*
5.0
-*
5.4
-*
5.9
-*
6.6
6.8
-*
6.7
6.4
-*
-*
5.9
-*
-*
-*
-*
5.2
6.0
5.8
-*
6.8
6.8
6.8
6.2
6.3
-*
6.7
6.2
6
6.5
6.4
6.0
5.3
5.9
6.3
5.5
5.8
6.9
5.9
6.6
6.2
6.0
7.0
3.9
4.1
4.1
-*
4.0
-*
4
-*
-*
-*
3.8
-*
2.9
3.7
2.9
-*
-*
3.7
-*
3.9
3.9
3.9
4.3
-*
-*
2
3
4
5
6
7
8
9
10
11
12
13
15
16
17
20
21
22
23
24
25
26
27
5.9
5.1
5.6
5.3
5.9
5.9
3.4
5.9
5.8
4.5
-*
4.3
-*
-*
-*
-*
5.8
6.6
6.1
5.8
6.6
5.9
6.2
*-This target is not included in top 300 target of this compound.
8

2.3.Biological activity
2.3.1. in vitro anti proliferative activity
Twenty three compounds 1-13, 15-17 and 23-27 were selected to be screened for their
anticancer activity in vitro against five representative cell lines of hepatocellular
carcinoma (liver) HepG-2, epdermoid carcinoma (larynx) Hep-2, mammary gland
(breast) MCF-7, human prostate cancer PC-3 and epitheloid cervix carcinoma HeLa
using the standard MTT method[41] The cell lines were obtained from ATCC via Holding
company for biological products and vaccines (VACSERA), Cairo, Egypt. Doxorubicin
was used as standard anticancer drug for comparison. The results of cytotoxic activity is
reported in Table 2.
MTT assay is a standard colorimetric assay for measuring cell growth. It is used to
determine cytotoxicity of potential medicinal agents and other toxic materials. In brief,
yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is reduced
to purple formazan by mitochondrial dehydrogenases of living cells. A suitable solvent is
added to dissolve the insoluble purple formazan product into a colored solution. The
absorbance of this colored solution can be quantified by measuring at a certain
wavelength. When the amount of purple formazan produced by cells treated with an
agent is compared with that produced by unreacted control cells, the effectiveness of the
agent in causing death of cells can deduced, through the production of a dose response
curve[42]
.
The obtained results showed variable degree of inhibitory activity towards the five tested
human tumor cell lines. Six compounds 15, 16, 17, 22, 24 and 25 among the tested
compounds showed good inhibitory activity towards all tested cell lines. (The dose
response curves of the six active compounds and doxorubicin are reported in Fig.1) As
for activity against hepatocellular carcinoma HepG-2 and mammary gland (breast)
MCF-7 cell line the highest activity was displayed by compounds 22 and 24 which
showed the percentage viability IC50 at 10.07 and 7.99 µM respectively for hepatocellular
carcinoma cell line HepG-2 while in case of mammary gland (breast) MCF-7 cell line
IC50 appeared at 7.17 and 6.84 µM respectively .
9

Further interpretation of the results revealed that in the epdermoid carcinoma (larynx)
Hep-2 , human prostate cancer PC-3 and epitheloid cervix carcinoma HeLa cell lines
compound 24 showed the highest activity with IC50 at 5.40 µM, 7.71 µM and 4.30 µM
respectively, being more potent than doxorubicin which showed IC50 at 6.32 µM, 8.87
µM and 5.57 µM respectively. Compounds 17 and 22 also showed remarkable inhibitory
activity against the three cell lines.
10

Table 2: Cytotoxicity (IC50) of tested compounds on different cell lines.
compound
In vitro cytotoxicity IC50 (µM)*
HepG-2
Hep-2
MCF-7
PC-3
HeLa
DOX
2
4.50±0.2
91.63±5.0
52.89±3.7
74.24±4.3
>100
6.32±0.3
89.69±4.9
42.35±2.8
86.52±4.5
97.60±5.3
51.14±3.2
81.23±4.4
71.14±3.7
88.96±4.8
76.93±3.9
57.55±3.4
46.84±3.0
73.21±3.8
15.88±1.3
19.87±1.5
9.58±0.7
4.17±0.2
82.14±4.7
53.42±3.3
68.90±4.1
94.17±5.0
38.19±2.6
72.18±4.2
47.71±3.0
77.54±4.5
63.40±3.8
56.42±3.5
64.54±3.9
45.95±2.8
8.16±0.8
8.87±0.6
>100
5.57±0.4
87.60±4.8
27.47±2.0
72.92±4.3
>100
3
41.48±2.7
>100
4
5
>100
6
38.47±2.7
78.35±4.5
51.18±3.4
83.00±4.7
64.10±4.0
58.77±3.8
70.77±4.1
45.78±3.1
14.22±1.3
23.57±1.8
15.91±1.4
35.70±2.5
41.48±2.9
10.07±1.0
32.75±2.3
7.99±0.6
42.92±2.8
87.36±4.8
64.71±4.0
>100
31.13±2.4
67.08±3.9
46.67±3.1
73.91±4.5
56.59±3.5
39.03±2.5
29.82±2.2
55.54±3.3
11.53±1.1
12.62±1.2
8.03±0.6
19.93±1.7
40.82±2.7
9.31±0.9
22.72±1.8
4.30±0.3
12.88±1.1
15.41±1.3
17.75±1.5
7
8
9
10
11
12
13
15
16
17
20
21
22
23
24
25
26
27
71.81±4.5
55.94±3.7
64.70±4.4
52.24±3.5
12.19±1.1
15.32±1.3
9.05±0.9
36.35±2.4
48.72±3.2
10.92±1.1
28.95±2.2
7.71±0.5
16.38±1.5
18.43±1.6
27.71±1.9
18.85±1.5
14.20±1.2
37.26±2.3
41.59±2.6
7.17±0.6
33.31±2.4
65.01±3.4
11.29±1.1
39.50±2.6
5.40±0.3
28.90±2.0
6.84±0.4
20.58±1.7
26.42±1.9
30.06±2.1
22.05±1.6
25.73±1.8
31.33±2.3
17.14±1.4
24.02±1.7
25.84±1.9
* IC50 (µM) : 1 – 10 (very strong). 11 – 20 (strong). 21 – 50 (moderate). 51 – 100 (weak) and above 100 (non-
cytotoxic) , DOX : Doxorubicin
11

1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
H eP G -2
H E P -2
M C F -7
P C -3
H eP G -2
H E P -2
M C F -7
P C -3
H eP G -2
H E P -2
M C F -7
P C -3
H ela
H ela
H ela
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
C o n cen tratio n o f 17 (µM )
C o n cen tratio n o f 16 (µM )
C o n cen tratio n o f 15 (µM )
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
H eP G -2
H E P -2
M C F -7
P C -3
H eP G -2
H E P -2
M C F -7
P C -3
H eP G -2
H E P -2
M C F -7
P C -3
H ela
H ela
H ela
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
C o n cen tratio n o f 22 (µM )
0
10
20
30
40
50
60
70
80
90
100
C o n cen tratio n o f 24 (µM )
C o n cen tratio n o f 27 (µM )
1 2 0
1 0 0
8 0
6 0
4 0
2 0
0
H eP G -2
H E P -2
M C F -7
P C -3
H ela
0
10
20
30
40
50
60
70
80
90
100
C o n cen tratio n o f D O X (µM )
Fig.1 : The dose response curves of the six active compounds and doxirubicin.
12

2.3.2. DNA/ methyl green assay
The mechanism of many antitumor agents involves binding with DNA. A variety of
methods have been utilized for the interaction of small molecular weight compounds with
DNA as DNA binding assay and methyl green binding assay. So, compounds 15, 16, 17,
24 and 25 were selected to test their DNA binding affinity compared to doxorubicin as
positive control.
DNA/methyl green assay is a colorimetric microassay method for the detection of DNA
binding agents. The methyl green dye reversibly binds DNA to form colored
DNA/methyl green complex. This color is stable at neutral pH. Upon displacement of
methyl green from DNA by DNA binding compounds, facilitates the addition of H2O
molecule to the dye, which results in formation of the colorless carbinol, followed by
spectrophotometrically a decrease in absorbance at 630 nm[43]. Results from DNA
binding assay (Table 3) revealed that compounds 15, 16 and 17 showed the highest
affinity for DNA, which was demonstrated by measuring IC50 (concentration required for
50% decrease in the initial absorbance of the DNA/methyl green solution). While
compounds 24 and 25 showed moderate and weak DNA binding affinity respectively.
Table 3: DNA/methyl green colorimetric assay of the tested compounds
Compounds. NO.
DNA/methyl green (IC50 µM)*
15
16
36.06±2.1
33.98±1.8
27.31±1.4
42.72±2.5
51.40±3.2
32.35±1.6
17
24
25
*DOX
IC50values represent the concentration (mean ± SD, n = 3–5 separate determina-
tions) required for a 50% decrease in the initial absorbance of the DNA/methyl-
green solution.
*DOX: Doxorubicin
13

2.3.3. Enzyme assay
In order to explain the observed anti proliferative activity and based on the pharmmapper
results, three enzymes; DNA polymerase, thymidylate synthase and tyrosine kinase were
selected to screen the enzyme inhibition activity of the synthesized compounds. These
enzymes are known to be targets of anticancer chemotherapeutic agents[44-48]
.
Five compounds with the highest antitumor activities( 15,16,17,24 and 25) were selected
to be evaluated against human DNA polymerase beta assay kit, human thymidylate
synthase ELISA kit and protein tyrosine kinase kit. The enzyme inhibition percentages
and IC50 values were calculated at serial dilutions of the tested compounds.(Table 4 and
5).
The tested compounds showed variable enzyme inhibition percentages for the three
enzymes. The results showed that the highest inhibition percentages were for DNA
polymerase ranging between 80 to 92% followed by thymidylate synthase with inhibition
percentages ranging between 75-92%. On other hand, tyrosine kinase showed the lowest
inhibition percentages ranging between 71-85%.
Compounds 16 (IC50-1.42µM) and 17 (IC50-0.9µM ) showed the highest inhibitory
activity against DNA polymerase compared to other tested compounds, while compounds
17 (IC50-27.97 µM) and 25 (IC50-29.19 µM) showed the highest inhibitory activity
against thymidylate synthase. Meanwhile, compounds 17 (IC50-73.4µM) and 24 (IC50-
135.5µM) were the most potent inhibitors for tyrosine kinase.
14

Table 4: Enzyme inhibition percentages of serial dilutions of the tested compounds against DNA
polymerase, thymidylate synthase and tyrosine kinase.
Enzyme inhibition percentage
Compound
NO.
Log conc.
DNA
polymerase
Thymidylate
synthase
Tyrosine
kinase
3
2
1
0
-1
3
2
1
0
-1
3
2
1
0
-1
3
2
1
0
-1
80.28273
65.94021
56.25092
12.57547
NT
92.32219
82.16757
69.63628
42.74775
NT
90.32838
68.75276
61.87601
52.54013
NT
85.57503
74.61346
63.95229
27.35974
NT
68.18881
35.02651
18.76176
0.803831
NT
84.37489
53.06995
28.40773
1.060373
NT
90.36942
74.10638
38.41286
3.43766
NT
75.88507
36.46314
16.82914
0.410467
NT
NT
15
16
17
24
25
71.77871
25.56022
9.803922
1.68067
NT
73.65546
28.5014
13.23529
0.07003
NT
85.07003
53.0042
17.507
3.361345
NT
79.90196
37.87115
15.68627
1.330532
3
2
1
0
-1
83.91989
65.94021
57.42895
34.63407
NT
92.53805
67.16265
42.55174
1.864204
NT
NT
77.03081
30.60924
11.69468
1.2605
NT : this compound is not tested at this concentration.
Table 5 :IC50 values of the tested compounds against DNA polymerase, thymidylate synthase and
tyrosine kinase.
Compound NO.
Enzymatic IC50 (µM)
DNA polymerase Thymidylate synthase
Tyrosine kinase
21.04
1.42
0.9
6.38
6.75
242.04
63.26
27.96
164.2
29.19
316.8
248.5
73.4
135.5
202.7
15
16
17
24
25
15

2.2.4.Structure activity relationship (SAR)
The replacement of chloroacetamido group in compound 2 with chloropropanamido
group in compound 3 enhanced the antitumor activity against all tested cell lines and
therefore the activity of compounds 11, 12 and 13 is much better than compounds 8, 9
and 10 .The para substitution on the phenyl moiety with floro or methyl group enhanced
the antitumor activity as seen in compounds 4 and 6 respectively, meanwhile, the para
substitution of phenyl with methoxy group greatly reduced the antitumor activity as seen
in compound 5. Also, the isosteric substitution of phenyl ring of compound 4 with
thiophene ring in compound 7 greatly reduced the activity.
The cyclization of o-amino ester thiophene into thienopyrimidines greatly enhanced the
activity as seen in compounds 15-27.The substitution of phenyl ring at N-3 of
thienopyrimidine ring with benzyl moiety in compound 17 enhanced the antitumor
activity. The S,N-dimethylation of compound 15 highly enhanced the antitumor activity
as seen in compound 22.
Regarding chalcones (23-27), replacement of para methoxy phenyl group in compound
23 with p-tolyl group in compound 24 or with 3,4,5-trimethoxy phenyl moiety in
compound 25 greatly enhanced the antitumor activity. On the other hand, the isosteric
replacement of phenyl moiety in compounds (23-25) with thiophene moiety in
compound 26 or with 4-bromo thiophene moiety in compound 27 reduced the activty
against the tested cell lines.
3.Conclusion
New thiophene and thienopyrimidine derivatives have been synthesized and their
antitumor activity and enzyme inhibitory activity against DNA polymerase, thymidylate
synthase and tyrosine kinase have been evaluated. Among the tested compounds,
15,16,17,24 and 25 were identified as the most active antitumor agents against the five
cell lines tested comparable to doxorubicin. Moreover, compound 17 was identified as
the most potent inhibitor for the three tested enzymes with IC50 0.9, 27.9 and 73.4 µM
respectively. Also, compounds 16,24 and 25 showed high enzyme inhibition percentages
16

against the screened enzymes .So, this study revealed that compounds 16,17,24 and 25
could be promising leads for further development of more potent antitumor agents.
4.Experimental
4.1.General
Ethyl 5-acetyl-2-amino-4-methylthiophene-3-carboxylate 1 was prepared following the
procedure reported by Abu-Hashem[24]. All the reagents and solvents were obtained from
commercial suppliers and used without any purification. The reactions were monitored by
using Fluka silica gel TLC alumminium cards (0.2 mm). Compounds were visualized by
using 254 nm UV lamp. Melting points (°C) were recorded using a Stuart digital melting
point apparatus SMP30 and are uncorrected. The IR spectra (KBr) were recorded on
Mattson 5000 FT IR spectrophotometer in (ν cm-1) in Central Laboratory Unit, Faculty of
Pharmacy, Mansoura University. 1H NMR , 13C NMR and 15N NMR were recorded on
Burker 400 MHz FT NMR spectrometer by using (DMSO-d6) or (CDCl3) as solvents
with chemichal shift being expressed in δ (ppm) with tetramethylsilane (TMS) as
standard. The mass spectrum was carried out on direct probe controller inlet part to single
quadropole mass analyzer in (thermo scientific GCMS) model (ISQ LT) using thermo x-
calibur software at the Regional Center for Mycology and Biotechnology (RCMB), Al-
Azhar University, Cairo. The enzyme inhibition assay was performed in confirmatory
diagnostic unit, VACSERA-EGYPT.
4.2. General procedure for the synthesis of compound (2 and 3)
A mixture of aminothiophene 1 (2.27gm, 10 mmol) and K2CO3(1.38 gm, 10 mmol) in
DMF (20 mL) was stirred in ice bath for 10 minutes. Then (30 mmol ) chloroacetyl
chloride or chloropropionyl chloride was added dropwise to the mixture, which is left
stirring at room temperature overnight. The mixture was then poured into crushed ice.
The formed yellowish white precipitate was separated by filtration, washed several times
with water and crystallized from aqueous ethanol.
4.2.1. Ethyl 5-acetyl-2-(2-chloroacetamido)-4-methylthiophene-3-carboxylate(2)
17

Yield:80% ; mp: 170 ˚C ; 1H NMR (δ, ppm, CDCl3): 1.4(t, 3H, J=13.6, CH3CH2COO-),
2.5 (s, 3H, CH3CO-), 2.7(s, 3H, CH3-thiophene ), 4.1(s, 2H, -Cl-CH2-CONH-), 4.4 (q,
2H, CH3CH2COO-), 12.4 (s, 1H, D2O exchangeable, NH-CO-). 13C NMR (δ, ppm,
CDCl3) :14.2 (CH3 ester), 16 (CH3 thiophene ), 30 (CH3 acetyl ), 42.2 (-CH2-CONH-),
61.6 (CH3CH2COO-), 116.1, 127.6, 143.6, 150.3 (thiophene quaternary carbons ), 164.5
(COO-), 166 (-CONH-), 191.5 (COCH3). MS m/z (%) Calcd for C12H14ClNO4S; 303.76,
found; 303 (2.9,M+).
4.2.2. Ethyl 5-acetyl-2-(3-chloropropanamido)-4-methylthiophene-3-carboxylate (3)
Yield:70% ; mp:108 ˚C ; IR (KBr, ν, cm-1) : 3252 ( NH amide ), 1716 (C=O acetyl ),
1667 (C=O amide ).1H NMR (δ, ppm, CDCl3): 1.4(t, 3H, J=13.6, CH3CH2COO-), 2.5 (s,
3H, CH3CO-), 2.7(s, 3H, CH3-thiophene ), 3.0(t, 2H, J=11.6, -CH2-CH2-CONH-), 3.9 (t,
2H, J=11.6, Cl-CH2-CH2-CONH-), 4.4 (q, 2H, CH3CH2COO-), 11.7 (s, 1H, D2O
exchangeable, NH-CO-). 13C NMR (δ, ppm, CDCl3) :14.2 (CH3 ester), 16 (CH3
thiophene ),30 (CH3 acetyl ),38 (-CH2-CONH-),39 (Cl-CH2-),61 (CH3CH2COO-
),115,127,143,151 (thiophene quaternary carbons ), 166 (COO-), 167 (-CONH-), 191
(COCH3). MS m/z (%) Calcd for C13H16ClNO4S; 317.78, found; 319 (66.05,M+2), 317
(100,M+).
4.3.General procedure for the synthesis of Ethyl 5-acetyl-4-methyl-2-(4-substituted
benzamido)thiophene-3-carboxylate (4-6)
A mixture of aminothiophene 1 (2.27gm,10 mmol ), triethyl amine (0.3 mL, 3mmol) and
the appropriate 4-substituted benzoyl chloride (40 mmol ) in chloroform (15 mL ) was
stirred at room temperature for 24 h. Then the reaction mixture was distilled under
reduced pressure and the residue obtained was washed several times with diluted aqueous
ammonia solution then crystallized from methanol.
4.3.1. Ethyl 5-acetyl-2-(4-fluorobenzamido)-4-methylthiophene-3-carboxylate(4).
Yield:70% ; mp:168 ˚C ;1H NMR (δ, ppm, CDCl3 ) :1.4 (t, 3H, J=14.4, CH3CH2COO)
,2.5 (s,3H,CH3CO-) ,2.8 (s,3H,CH3-thiophene), 4.4 (q, 2H, CH3CH2COO-), 7.2 (ddd, 2H
,Ar-H), 8.0 (ddd, 2H, Ar-H), 12.6 (s, 1H, D2O exchangable-CONH-) .13C NMR (δ, ppm,
18

CDCl3): 14.2 (CH3 ester ), 16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 61 (CH3CH2COO-),
115,127,143.8,152 (thiophene quaternary carbons ),116.2,116.4 ,128, 130.1 130.2 ,163
(phenyl aromatic carbons ), 164 (-CONH-), 167 (-COO-), 191 (CH3CO-). MS m/z(%):
Calcd for C17H16FNO4S; 349.38, found; 349 (100, M+).
4.3.2. Ethyl 5-acetyl-2-(4-methoxybenzamido)-4-methylthiophene-3-carboxylate (5).
Yield:45% ; mp:205˚C ;1H NMR (δ, ppm, CDCl3 ): 1.4 (t, 3H, J=14.4, CH3CH2COO),
2.5 (s, 3H, CH3CO-), 2.8 (s, 3H, CH3-thiophene), 3.9 (s, 3H, -OCH3), 4.4 (q, 2H,
CH3CH2COO-), 7.0 (d, 2H, J=8.8, Ar-H), 8.0 (d,2H, J=8.4, Ar-H), 12.5 (s,1H, D2O
exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3): 14.2 (CH3 ester ), 16.15 (CH3
thiophene ), 30 (CH3 acetyl ), 55 (-OCH3) ,61(CH3CH2COO-) , 114.8 ,127, 143 ,152.7
(Thiophene quaternary carbons) , 114.3, 123.9, 129.6, 163.5 (phenyl aromatic carbons)
,163 (-CONH-), 167 (-COO-), 191 (CH3CO-). MS m/z (%): Calcd for C18H19NO5S;
361.41, found; 361(100, M+).
4.3.3. Ethyl 5-acetyl-4-methyl-2-(4-methylbenzamido)thiophene-3-carboxylate (6).
Yield:65% ; mp:159˚C ;1H NMR (δ, ppm, CDCl3 ): 1.4 (t, 3H, J=14.4, CH3CH2COO)
,2.47 (s, 3H, CH3CO-), 2.5 (s, 3H, CH3-thiophene), 2.8 (s, 3H, -CH3-phenyl) ,4.4 (q, 2H,
CH3CH2COO-), 7.3(d, 2H, J=8, Ar-H), 7.9 (d, 2H , J=8.4, Ar-H), 12.6 (s, 1H, D2O
exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3) : 14.2 (CH3 ester ), 16.15 (CH3
thiophene ), 21.7 (CH3-phenyl), 30 (CH3 acetyl ), 61(CH3CH2COO-), 115 ,126.8, 143
,152 (thiophene quaternary carbons), 127.6 ,128.8 ,129.1 ,129.8 (phenyl aromatic
carbons),164.2 (-CONH-) ,167.13 (-COO-) ,191.17 (CH3CO-). MS m/z (%):Calcd for
C18H19NO4S; 345.41, found; 345(100, M+).
4.4. Synthesis of ethyl 5-acetyl-4-methyl-2-(thiophene-2-carboxamido)thiophene-3-
carboxylate (7)
A mixture of aminothiophene 1 (2.27gm, 10 mmol ), triethyl amine (0.3 mL, 3mmol )
and 2-thiophene carbonyl chloride (2.9gm, 20 mmol ) in chloroform (15 mL ) was stirred
at room temperature for 24 h. Then the reaction mixture was distilled under reduced
pressure and the residue obtained was washed several times with diluted aqueous
ammonia solution then crystallized from methanol.
19

Yield: 80% ; mp:158◦C ; 1H NMR (δ, ppm, CDCl3 ): 1.4 (t, 3H, J=14.4, CH3CH2COO),
2.5 (s, 3H, CH3CO-), 2.8 (s, 3H, CH3-thiophene), 4.4 (q, 2H, CH3CH2COO-), 7.2 (t, 1H
,J=8.8 ,Ar-H), 7.7 (d, 1H, J=4.8, Ar-H), 7.8 (d, 1H, J=3.6, Ar-H ) 12.5 (s, 1H, D2O
exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3): 14.2 (CH3 ester ), 16.15 (CH3
thiophene ), 30 (CH3 acetyl ), 61(CH3CH2COO-) , 115 ,127, 143.9,152 (thiophene
quaternary carbons), 128,130,132,136.7 (thiophene carbons), 159 (-CONH-), 167 (-COO-
), 191.6 (CH3CO-). MS m/z (%):Calcd for C15H15NO4S2;337.41, found; 337(21.2, M+).
4.5.General procedure for synthesis of compounds (8-13)
A mixture of compound 2 or compound 3 (10 mmol), triethyl amine (0.3mL, 3 mmol
) and appropriate 2ry aliphatic amine (10 mmol ) in DMF (10 mL ) was refluxed for 8 h.
The reaction mixture was cooled and poured into crushed ice and the formed precipitate
was filtered, washed with water and crystallized from ethanol.
4.5.1. Ethyl 5-acetyl-4-methyl-2-(2-(4-phenylpiperazin-1-yl)acetamido)thiophene-3-
carboxylate (8).
Yield:50% ; mp:189◦C ; IR (KBr, ν, cm-1) : 3440-3447 ( NH amide ), 1709-1716 (C=O
acetyl ), 1740 (C=O ester), 1660-1669 (C=Oamide ).1H NMR (δ, ppm, CDCl3 ): 1.3 (t
,3H , J=13.6, CH3CH2COO), 2.4 (s, 3H, CH3CO-), 2.7(s, 3H, CH3-thiophene), 2.76 (br s,
4H, piperazine ), 3.2 (br s, 4H, piperazine ), 3.3 (s, 2H, -NHCOCH2- ), 4.3 (q, 2H,
CH3CH2COO-), 6.8 (t, 1H, J=13.6, Ar-p-H), 6.9 (d, 2H, J=7.6, Ar-H), 7.2 (m, 2H, Ar-H )
12.6 (s, 1H, D2O exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3) : 14.2 (CH3 ester ),
16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 49, 53 (carbons of piperazine ring ), 61 (-
NHCOCH2-) 61.2 (CH3CH2COO-), 116, 127, 143 ,150 ( thiophene quaternary carbons),
115,8, 129.32 (aromatic phenyl carbons ),165.22 (-CONH-), 169 (-COO-), 191.7
(CH3CO-). MS m/z (%): Calcd for C22H27N3O4S; 329.53, found; 429(100, M+).
4.5.2. Ethyl 5-acetyl-2-(2-(4-benzylpiperazin-1-yl)acetamido)-4-methylthiophene-3-
carboxylate (9).
Yield:77% ; mp:183◦C ; IR (KBr, ν,cm-1) : 3440-3447 ( NH amide ), 1709-1716 (C=O
acetyl ), 1740 (C=O ester), 1660-1669 (C=O amide ).1H NMR (δ, ppm, CDCl3 ): 1.4 (t,
3H, J=14, CH3CH2COO), 2.5 (s, 3H, CH3CO-), 2.7(s, 3H, CH3-thiophene), 2.6 (m, 8H,
20

piperazine ), 3.3 (s, 2H, -NHCOCH2- ), 3.6 (s, 2H, -CH2-aromatic ), 4.4 (q, 2H,
CH3CH2COO-), 6.8 (t, 1H, J=13.6, Ar-p-H), 6.9 (d, 2H , J=7.6, Ar-H), 7.3-7.4(m, 5H,
Ar-H ) 12.5 (s, 1H, D2O exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3) : 14.3 (CH3
ester ), 16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 52 , 53 (carbons of piperazine ring ),
61.1 (-NHCOCH2-), 61.1(CH3CH2COO-), 62.8 (-CH2-aromatic ), 115 ,127, 143 ,150 (
thiophene quaternary carbons), 126,128,129.2,137.7 (aromatic phenyl carbons ),165 (-
CONH-) ,169.5 (-COO-), 191.7 (CH3CO-). MS m/z (%): Calcd for C23H29N3O3S; 443.56,
found; 443(100, M+).
4.5.3. Ethyl 5-acetyl-4-methyl-2-(2-morpholinoacetamido)thiophene-3-carboxylate (10).
Yield:66% ; mp:190◦C; 1H NMR (δ, ppm, CDCl3 ): 1.4 (t , 3H , J=14,CH3CH2COO), 2.5
(s, 3H, CH3CO-), 2.7(s, 3H, CH3-thiophene), 2.6 (t, 4H, J=9.2, (CH 2)2N-morpholine ),
3.3 (s, 2H, -NHCOCH2- ), 3.8 (t, 4H, J=9.2, (CH 2)2O-morpholine ), 4.4 (q, 2H,
CH3CH2COO-), 12.5 (s, 1H, D2O exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3):
14.3 (CH3 ester ), 16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 53, 66(carbons of
morpholine ring ), 61.1 (-NHCOCH2-) 61.3(CH3CH2COO-) , 115 ,127, 143 ,150
(thiophene quaternary carbons) ,169 (-CONH-),171 (-COO-), 191 (CH3CO-). MS m/z
(%): Calcd for C16H22N2O5S; 354.42, found; 454(100, M+).
4.5.4. Ethyl 5-acetyl-4-methyl-2-(3-(4-phenylpiperazin-1-yl)propanamido)thiophene-3-
carboxylate (11).
Yield:40% ; mp:108◦C; IR (KBr, ν ,cm-1) : 3440-3447 ( NH amide ), 1709-1716 (C=O
acetyl ), 1740 (C=O ester), 1660-1669 (C=O amide ).1H NMR (δ, ppm, CDCl3 ): 1.3 (t ,
3H, J=14, CH3CH2COO), 2.5 (s, 3H, CH3CO-), 2.72 (m, 4H, piperazine ) 2.76(s, 3H,
CH3-thiophene), 2.8 (t, 4H, piperazine ), 3.3 (t, 4H , J=9.6, -NHCO-CH2-CH2- ), 4.3 (q ,
2H, CH3CH2COO-), 6.8 (t, 1H ,J=14.4, Ar-p-H), 6.9 (d, 2H, J=8.4, Ar-H), 7.2 (t, 2H,
J=15.2, Ar-H ), 12.3 (s, 1H, D2O exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3) :
14 (CH3 ester), 16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 33 (- CH2-CONH-), 45 (CH2-
CH2-CONH-), 48, 53 (carbons of piperazine ring ), 61 (-NHCOCH2-), 115 ,127, 143
,151.3(thiophene quaternary carbons), 116, 119.72, 129, 151.73 (aromatic phenyl carbons
21

), 165.8 (-CONH-), 170 (-COO-), 191.8 (CH3CO-). MS m/z (%): Calcd for C23H29N3O4S;
443.56, found; 443(100, M+).
4.5.5. Ethyl 5-acetyl-2-(3-(4-benzylpiperazin-1-yl)propanamido)-4-methylthiophene-3-
carboxylate (12).
Yield:64% ; mp:112◦C; IR (KBr, ν, cm-1) : 3440-3447 ( NH amide ), 1709-1716 (C=O
acetyl ), 1740 (C=O ester) ,1660-1669 (C=O amide ) .1H NMR (δ, ppm, CDCl3 ): 1.3 (t,
3H, J=14, CH3CH2COO), 2.45 (s, 3H, CH3CO-), 2.5 (br s, 8 H, piperazine ), 2.6 (t, 2H,
CH2-CH2-CONH-), 2.7(s, 3H, CH3-thiophene), 2.9 (t, 2H, (- CH2-CONH-), 3.4 (s, 2H,-
CH2-aromatic ), 4.3 (q, 2H, CH3CH2COO-), 7.2-7.3 (m, 5H, Ar-H ), 12.2 (s, 1H, D2O
exchangeable -CONH-). 13C NMR (δ, ppm, CDCl3) : 14 (CH3 ester ), 16.15 (CH3
thiophene ), 30 (CH3 acetyl ), 33 (-CONH- CH2-CH2), 52.4 (-CONH-CH2-CH2-), 53,55,
(carbons of piperazine ring ) ,61(CH3CH2COO-),63.1 (-CH2-aromatic ), 115,138, 143
,151(thiophene quaternary carbons), 126.7,127.1,128.2,129 (aromatic phenyl carbons ),
165 (-CONH-), 170.4 (-COO-), 191.8 (CH3CO-). MS m/z (%): Calcd for C24H31N3O4S;
457.59, found; 457(100, M+).
4.5.6. Ethyl 5-acetyl-4-methyl-2-(3-morpholinopropanamido)thiophene-3-carboxylate
(13).
Yield:73% ; mp:122◦C; IR (KBr, ν, cm-1) : 3440-3447 ( NH amide ), 1709-1716 (C=O
acetyl ), 1740 (C=O ester), 1660-1669 (C=O amide ).1H NMR (δ, ppm, CDCl3 ): 1.3 (t
,3H , J=14, CH3CH2COO), 2.45 (s, 3H, CH3CO-), 2.5-2.68(m, 8H, -CH2-CH2- + (CH
2)2N-morpholine ), 2.7 (s, 3H, CH3-thiophene), 3.7 (t, 4H, J=9.2, (CH 2)2O-morpholine )
,4.4 (q, 2H, CH3CH2COO-), 12.2 (s, 1H, D2O exchangeable -CONH-). 13C NMR (δ,
ppm, CDCl3) : 14.3 (CH3 ester ), 16.15 (CH3 thiophene ), 30 (CH3 acetyl ), 33 (-CONH-
CH2-CH2), 53 (-CONH-CH2-CH2-), 53.51 ,66.5 (carbons of morpholine ring ), 61.1 (-
NHCOCH2-), 61.3(CH3CH2COO-), 115,126, 143,151 (thiophene quaternary carbons)
,165 (-CONH-), 170 (-COO-), 191.8 (CH3CO-). MS m/z (%):Calcd for C17H24N2O5S;
368.45, found; 368(100, M+).
22

4.6.Synthesis of ethyl 5-acetyl-2-hydrazinyl-4-methylthiophene-3-carboxylate (14).
A mixture of compound 2 or compound 3 (10 mmol) and hydrazine hydrate (1.5gm, 30
mmol ) in ethanol (10 mL ) was refluxed for 4 h. The reaction mixture was cooled,
poured into crushed ice and left overnight. The obtained crystals were filtered, washed
with water and crystallized from methanol.
Yield:30% ; mp:237◦C; IR (KBr, ν, cm-1) : 3415-3303 cm-1 (NHNH2), 1700 cm-1
(COCH3), 1744 cm-1 (-COO-). 1H NMR (δ, ppm, CDCl3): 1.3 (t, 3H, J=14,
CH3CH2COO), 2.15(s, 3H, CH3CO-), 2.4(s, 3H, CH3-thiophene), 4.4 (q, 2H,
CH3CH2COO-), 5.2 (s, 1H, D2O exchangeable, thiophene-NHNH2), 6.2 (s, 2H, D2O
exchangeable, thiophene-NHNH2). 13C NMR (δ, ppm, CDCl3) : 14.4 (CH3 ester ), 15.6
(CH3 thiophene ), 16.8 (CH3 acetyl ), 59.8(CH3CH2COO-) , 107.6 ,120, 133.1,144.79(
thiophene quaternary carbons) ,162.9(-COO-) , 166.1 (CH3CO-). N15NMR : 2 nitrogen
atoms at 200 and 406 ppm. MS m/z (%): calc for C10H14N2O3S; 242.29, found; 242.06
(3.94, M+).
4.7.General procedure for synthesis of compounds (15-17)
An equimolar mixture of aminothiophene 1 (2.27 gm, 10mmol), NaOH (0.4gm, 10mmol
) and the appropriate isothiocyanate derivative (10 mmol) in DMF (20 mL ) was refluxed
for 6 h. The reaction mixture was cooled, poured into crushed ice, neutralized with
diluted acetic acid and the solid formed was filtered and crystallized from ethanol.
4.7.1. 6-Acetyl-2-mercapto-5-methyl-3-phenylthieno[2,3-d]pyrimidin-4(3H)-one (15).
Yield:80% ; mp:246◦C; IR ( KBr, ν, cm-1) :3394 (NH amide), 1669 (C=O amide), 1236
(C=S).1H NMR (δ, ppm, DMSO-d6): 2.5(s, 3H, -COCH3), 2.7 (s, 3H, CH3-thiophene),
7.2 (d, 2H, Ar-H), 7.4 (m, 1H, Ar-H), 7.48 (t, 2H, Ar-H). 13C NMR (δ, ppm, DMSO-d6
):15 (CH3 thiophene ), 30 (CH3 acetyl ), 118, 128, 139, 154(thiophene quaternary
carbons), 129, 131.04, 142, 150 (phenyl ), 162 (C-SH), 176 (-CONH-),191(CH3CO-).
MS m/z (%): calcd for C15H12N2O2S2; 316.39, found; 316(23.4, M+).
23

4.7.2. 6-Acetyl-3-(4-chlorophenyl)-2-mercapto-5-methylthieno[2,3-d]pyrimidin-4(3H)-
one (16).
Yield:70% ; mp:159◦C; IR (KBr, ν, cm-1): 3453 (NH amide), 1677 (C=O amide), 1217
(C=S) .1H NMR ( δ, ppm, CDCl3): 2.6(s, 3H, -COCH3), 2.8 (s, 3H, CH3-thiophene), 7.19
(d, 2H, Ar-H),7.5 (d, 2H, Ar-H). 13C NMR (δ, ppm, DMSO-d6 ): 15 (CH3 thiophene ), 30
(CH3 acetyl ), 118, 135, 143,153 (thiophene quaternary carbons), 126, 129, 132,135
(phenyl ), 163 (C-SH), 176 (-CONH-), 191(CH3CO-). MS m/z (%): calcd for
C15H11ClN2O2S2 ; 350.84, found; 349.9(100, M+).
4.7.3. 6-Acetyl-3-benzyl-2-mercapto-5-methylthieno[2,3-d]pyrimidin-4(3H)-one (17).
Yield:75% ; mp:211◦C; IR (KBr,ν,cm-1) : 3449 (NH amide), 1695 (C=O amide), 1295
(C=S).1H NMR (δ, ppm, CDCl3): 2.5(s, 3H, -COCH3), 2.8 (s, 3H, CH3-thiophene), 5.7
(s, 2H, CH2-phenyl), 7.3-7.5 (m, 5H, Ar-H), 12.0 (s, 1H, -NH-). 13C NMR (δ, ppm,
DMSO-d6 ):15 (CH3 thiophene ), 30 (CH3 acetyl ), 117.9,135, 143,152 (thiophene
quaternary carbons) ,128,128.5,129,136 (phenyl ), 157 (C-SH) ,176 (-CONH-
),191(CH3CO-). MS m/z (%): calcd for C16H14N2O2S2; 330.4, found; 329.9(100, M+).
4.8.General procedure for synthesis of compounds (18 and 19).
A mixture of compound 15 or 17 (10 mmol) and hydrazine hydrate (5 mL) in
pyridine(10 mL) was refluxed for 14 h .The reaction mixture was cooled, poured into
crushed ice and the product was filtered and crystallized from ethanol.
4.8.1. 6-Acetyl-2-hydrazinyl-5-methyl-3-phenylthieno[2,3-d]pyrimidin-4(3H)-one (18).
Yield:39% ; mp: > 300 ◦C; IR (KBr, ν, cm-1): 3447-3382 (NHNH2).1H NMR ( δ, ppm,
DMSO-d6): 2.09 (s, 3H, -COCH3), 2.4 (s, 3H, CH3-thiophene), 6.3-6.8 (br s, 2H, -
NHNH2, D2O exchangeable), 7.19 (d, 2H, Ar-H), 7.4 (d, 1H, Ar-H), 7.5 (m, 3H, 2 Ar-H
+ -NHNH2). 13C NMR (δ, ppm, DMSO-d6 ): 15 (CH3 thiophene ), 30 (CH3 acetyl ), 118,
135, 143,153 (thiophene quaternary carbons), 126, 129 ,132, 135 (phenyl ), 176 (-
CONH), 191(CH3CO-). MS m/z (%): calcd for C15H14N4O2S; 314.36, found; 314 (18,
M+).
4.8.2. 6-Acetyl-3-benzyl-2-hydrazinyl-5-methylthieno[2,3-d]pyrimidin-4(3H)-one (19).
24

Yield:30% ; mp: > 300 ◦C; IR (KBr,ν,cm-1) :3387 (NHNH2). 1H NMR (δ, ppm, DMSO-
d6): 2.5(s, 3H, -COCH3), 2.8 (s, 3H, CH3-thiophene), 5.7 (s, 2H, CH2-phenyl), 7.3-7.5 (m,
5H, Ar-H).13C NMR (δ, ppm, DMSO-d6 ): 15 (CH3 thiophene ), 30 (CH3 acetyl ),
117.9,135, 143,152 (thiophene quaternary carbons), 128, 128.5, 129,136 (phenyl ), 176 (-
CONH-), 191(CH3CO-). MS m/z (%): calcd for C16H16N4O2S; 328.39, found; 330 (21.5,
M+).
4.9.Synthesis of ethyl 5-acetyl-(((dimethylamino)methylene)amino)-4-methylthiophene-
3-carboxylate (20).
A mixture of aminothiophene 1 (2.27gm,10 mmol) in DMFDMA (10 mL) was refluxed
for 2h. The reaction mixture was cooled and the precipitate formed was filtered, washed
and crystallized from ethanol.
Yield:53% ; mp: 99 ◦C;1H NMR (δ, ppm, CDCl3 ):1.3 (t, 3H, J=14.4, CH3CH2COO), 2.4
(s, 3H, CH3CO-), 2.6(s, 3H, CH3-thiophene), 3.3 (s, 6H, dimethyl amino-), 4.3 (q, 2H,
CH3CH2COO-), 7.7 (s, 1H ,-N=CH-).13C NMR (δ, ppm, CDCl3): 15 (CH3 ester ), 16.15
(CH3 thiophene ), 30 (CH3 acetyl ), 40 (dimethyl amino-), 61 (CH3CH2COO-) ,
111,127,143,153 (thiophene quaternary carbons ), 164 (-N=CH-), 171 (-COO-),191
(CH3CO-). MS m/z (%): calcd for C13H18N2O3S; 282.1, found; 282 (15.5, M+).
4.10.Synthesis of ethyl 5-acetyl-2-formamido-4-methylthiophene-3-carboxylate(21).
Procedure A :A mixture of compound 20 (2.5gm,10mmol ) in glacial acetic acid (15 mL)
was refluxed for 1h.The reaction mixture was cooled and poured into crushed ice. The
precipitate was filtered and washed with water.
Procedure B : A mixture of aminothiophene 1 (2.27 g, 10 mmol) and ammonium acetate
(0.3 g, 3.89mmol ) in formic acid (20 mL ) was refluxed for 18 h .The reaction mixture
was cooled and neutralized by diluted aqueous ammonia solution. The precipitate formed
was filtered and crystallized from hexane.
Yield:63% ; mp: 107 ◦C;1H NMR (δ, ppm, CDCl3 ) :1.4 (t ,3H, J=14.4 ,CH3CH2COO-)
,2.5 (s,3H,CH3CO-), 2.7 (s, 3H, CH3-thiophene), 4.3 (q , 2H, CH3CH2COO-), 8.6 (s, 1H
,-NHCHO), 11.6 (s, 1H, -NHCHO). 13C NMR (δ, ppm, CDCl3) : 14.2 (CH3 ester ), 16
25

(CH3 thiophene ), 30 (CH3 acetyl ), 61 (CH3CH2COO-), 115,127.4,143.5,149.7
(thiophene quaternary carbons ),157 (-NHCHO) ,166 (-COO-), 191 (CH3CO-). MS m/z
(%): calcd for C11H13NO4S;255.29, found; 254.9 (12.9, M+).
4.11.Synthesis of 6-acetyl-1,5-dimethyl-2-(methylthio)-4-oxo-3-phenyl-3,4-
dihydrothieno[2,3-d]pyrimidin-1-ium (22).
A mixture of compound 15 (3.16gm,10 mmol ) in DMFDMA (20 mL) was refluxed for
3h.The reaction was cooled and poured into crushed ice. The yellow precipitate was
filtered and crystallized from ethanol.
Yield: 30% ; mp:101◦C;1H NMR (δ, ppm,CDCl3 ):1.27( s, 3H, -S-CH3), 2.5(s, 3H,-
COCH3), 2.7 (s, 3H, CH3-thiophene), 2.92 (s, 3H, -N-CH3), 7.3 (m, 2H, Ar-H), 7.58
(m,3H,Ar-H). 13C NMR (δ, ppm, CDCl3): 15.6 (-S-CH3), 15.7(CH3-thiophene), 30 (CH3
acetyl ), 30.6 (-N-CH3) 128, 137,143 ,154 ( thiophene quaternary carbons),120,129
,130,135 (phenyl ),162.3(C-SH) ,165(-CONH-),191.8(CH3CO-). MS m/z (%): calcd for
C17H17N2O2S2; 345.4, found; 345.9 (4.17, M+).
4.12.General procedure for synthesis of compounds (23-27).
Procedure A : A mixture of appropriate aldehyde (10 mmol ) and compound 15 (3.16gm,
10 mmol) was dissolved in (10 mL) absolute ethanol and stirred at room temperature for
15 minutes. Then 10 % NaOH solution was added dropwise to the reaction mixture. The
mixture was stirred for 6 h, neutralized by 0.1 N HCl whereby the precipitation occurred.
The precipitate formed was filtered, washed with water and crystallized from ethanol.
Procedure B : A mixture of appropriate aldehyde (10 mmol ) and compound 15 (3.16gm,
10 mmol) was dissolved in 15 mL sodium ethoxide solution (0.2 g of sodium metal in 15
mL absolute ethanol ) and stirred for 3h at room temperature. The precipitate formed was
filtered and crystallized from ethanol. There was no significant difference between the
two procedures in yield percentages.
4.12.1. 2-Mercapto-6-(3-(4-methoxyphenyl)acryloyl)-5-methyl-3-phenylthieno[2,3-
d]pyrimidin-4(3H)-one (23).
26

Yield: 72% ; mp:278◦C; 1H NMR (δ, ppm,CDCl3 ): 2.9 (s, 3H, CH3-thiophene), 3.8 (s,
3H, -O-CH3), 6.9 (d, 2H, J=8, Ar-H), 7.1 (d, 1H, J=15.2, -CH=CH-CO-), 7.3 (d, 1H, Ar-
H), 7.5-7.6 (m, 6H, Ar-H), 7.9 (d, 1H, J=15.2, -CH=CH-CO-). 13C NMR (δ,
ppm,CDCl3): 15.8(CH3-thiophene), 55 (OCH3)118.4, 127, 138.3,156 (thiophene
quaternary carbons), 129.1, 145.5 (2 alkene carbons ) 114.5, 120.6, 128.3 ,129.7, 130.6
(phenyl aromatic carbons ), 162.2(C-SH) ,176(-CONH-),183.8(-CH=CHCO-). MS m/z
(%): calcd for C23H18N2O3S2; 434.53, found; 434.04 (100, M+).
4.12.2. 2-Mercapto-5-methyl-3-phenyl-6-(3-(p-tolyl)acryloyl)thieno[2,3-d]pyrimidin-
4(3H)-one (24).
Yield: 59% ; mp:262◦C; 1H NMR (δ, ppm, DMSO-d6 ):2.3 (s, 3H, CH3-thiophene), 2.8
(s, 3H, CH3 -Phenyl), 7 (d, 2H, J=7.6, Ar-H), 7.2 (t, 3H, J=13.6, Ar-H), 7.35 (m, 2H, Ar-
H) , 7.4 (d, 1H, J=14.8, -CH=CH-CO-), 7.6 (d, 1H, J=15.2, -CH=CH-CO-), 7.7 (d, 2H,
J=8, Ar-H ).13C NMR (δ, ppm, DMSO-d6): 16.4(CH3-thiophene), 21.5 (phenyl-CH3)
,129.1 ,144.5 (2 alkene carbons )117.29,127, 132.3,142.4(thiophene quaternary carbons)
,124.5,126.1, 128.8 ,129.7 ,130 ,140.8 ,142.1(phenyl aromatic carbons ), 161.8(C-SH)
,180.3(-CONH-), 183.04(-CH=CHCO-). MS m/z (%): calcd for C23H18N2O2S2; 418.53,
found; 418.06(100, M+).
4.12.3. 2-Mercapto-5-methyl-3-phenyl-6-(3-(3,4,5-trimethoxyphenyl)acryloyl)thieno[2,3-
d]pyrimidin-4(3H)-one (25).
Yield: 76% ; mp:259◦C; 1H NMR (δ, ppm, DMSO-d6 ): 2.8 (s, 3H, CH3-Thiophene), 3.7
(s, 3H, p-OCH3), 3.8 (s, 6H, 2 m-OCH3), 7 (d, 2H, J=8, Ar-H), 7.1 (br s, 2H, 2 H of
trimethoxy phenyl ring), 7.2 (t, 1H, J=14.4, Ar-H), 7.3-7.4 (m, 3H, 2Ar-H & -CH=CH-
CO-), 7.5 (d, 1H, J=15.6, -CH=CH-CO-).13C NMR (δ, ppm, DMSO-d6): 16.4(CH3-
thiophene), 56.5 (2 m-OCH3), 60.6 (p-OCH3) 129.7, 144.5 (2 alkene carbons )
106,130.7,139.9, 142(trimethoxy phenyl aromatic carbons) ,125.2 ,126 ,128.8 ,126 ,139
(phenyl aromatic carbons ), 117,126.9,142.6 , 153.5 (thiophene quaternary aromatic
carbons), 161(C-SH), 168.8(-CONH-), 183.04(-CH=CHCO-). MS m/z (%):calcd for
C25H22N2O5S2; 494.58, found; 494.6(48.2, M+).
27

4.12.4. 2-Mercapto-5-methyl-3-phenyl-6-(3-(thiophen-2-yl)acryloyl)thieno[2,3-
d]pyrimidin-4(3H)-one (26).
Yield: 66% ; mp272◦C; 1H NMR (δ, ppm,CDCl3 ): 2.9 (s, 3H, CH3-thiophene),7.1 (d, 2H,
J=14.8, Ar-H), 7.4 (d, 1H, -CH=CH-CO-), 7.5-7.6 (m, 6H, Ar-H), 8 (d, 1H, J=15.2,-
CH=CH-CO-).13C
NMR
(δ,
ppm,CDCl3):
15.7(CH3-thiophene),
117,128,
137,145(thiophene quaternary carbons),129.1,143.13 (2 alkene carbons )121.9, 128.3
,139.8 ,128.5 (phenyl aromatic carbons ) ,139.8,129.7,132.9,128.3( thiophene carbons )
162(C-SH), 163(-CONH-),173(-CH=CHCO-). MS m/z (%): calcd for C20H14N2O2S3;
410.02, found; 409.97(100, M+).
4.12.5. 6-(3-(4-Bromothiophen-2-yl)acryloyl)-2-mercapto-5-methyl-3-phenylthieno[2,3-
d]pyrimidin-4(3H)-one (27).
Yield: 46% ; >300 mp◦C ; 1H NMR (δ, ppm, DMSO-d6 ): 2.1 (s, 3H, CH3-thiophene), 7.1
(d, 1H, J=11.6, -CH=CH-CO-), 7.3-7.5 (m, 7H, Ar-H), 7.8(d, 1H, J=15.2, -CH=CH-CO-
). 13C NMR (δ, ppm,DMSO-d6): 15 (CH3-thiophene), 117,129.6, 134,141.7 (thiophene
quaternary carbons) ,129.1,143.13 (2 alkene carbons )129.3 (phenyl aromatic carbons )
,86,128.3,130.5,132( bromo thiophene carbons ) 162(C-SH) ,163 (-CONH-),173(-
CH=CHCO-). MS m/z (%): calcd for C20H13BrN2O2S3; 489.42, found; 489.3(100, M+).
4.13.Target prediction
The target compounds 2-27 were uploaded in Tripos Mol2 format. PharmMapper finds
the best mapping poses of the uploaded molecule against all the targets in Pharm
Target DB and top N potential drug targets as well as respective molecule's aligned
poses are outputted.
4.14.In vitro antitumor screening
Materials and methods
Five human tumor cell line namely ; hepatocellular carcinoma (HepG-2) , Mammary
gland (MCF-7), epitheliod Carcinoma (HeLa) , epidermoid carcinoma (Hep2) and
Human prostate cancer (PC-3) were obtained from ATCC via Holding company for
biological products and vaccines (VACSERA), Cairo, Egypt. Doxorubicin was used as a
28

standard anticancer drug for comparison. The reagents used were RPMI-1640 medium ,
MTT and DMSO (sigma co., St. Louis, USA), Fetal Bovine serum (GIBCO, UK).
MTT procedure
The cell lines mentioned above were used to determine the inhibitory effects of
compounds on cell growth using the MTT assay. This colorimetric assay is based on the
conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative by
mitochondrial succinate dehydrogenase in viable cells. Cell lines were cultured in RPMI-
1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml
penicillin and 100µg/ml streptomycin at 37 C in a 5% Co2 incubator. The cell lines were
seeds in a 96-well plate at a density of 1.0x104 cells/well. at 37 C for 48 h under 5% Co2.
After incubation the cells were treated with different concentration of compounds and
incubated for 24 h. After 24 h of drug treatment, 20 µl of MTT solution at 5mg/ml was
added and incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 µl is added
into each well to dissolve the purple formazan formed. The colorimetric assay is
measured and recorded at absorbance of 570 nm using a plate reader (EXL 800 , USA).
The relative cell viability in percentage was calculated as (A570 of treated samples/A570
of untreated sample) X 100. The IC50 values were calculated according to the equation
for Boltzmann sigmoidal concentration response curve using the nonlinear regression
fitting models.
4.15.DNA/methyl green assay
DNA methyl green (20 mg) was suspended in 100 ml of 0.05 MTris-HCl buffer (pH 7.5)
containing 7.5 mM MgSO4; the mixture was stirred at 37 °C with a magnetic stirrer for
24 h. Test samples(10, 100, 1000 mg) were dissolved in ethanol in ependoff tubes,
solvent was removed under vacuum, and 200 mL of the DNA/methyl green solution were
added to each tube. Samples were incubated in the dark at ambient temperature. After 24
h, the final absorbance of the samples was determined at 642.5–645 nm. Readings were
corrected for initial absorbance and normalized as the as the percentage of the untreated
standard using ethidium bromide as positive control.
29